Clostridial toxin derivatives able to modify peripheral sensory afferent functions

ABSTRACT

This invention describes a novel agent for the targeted control of a mammalian cell activity, in particular the agent is used to control the interaction of particular cell types with their external environment. The agent has applications as a pharmaceutical for the treatment of a variety of disorders. An agent according to the invention comprises three Domains B, T and E linked together in the following manner: Domain B-Domain T-Domain E where Domain B is the Binding Domain which binds the agent to a Binding Site on the cell which undergoes endocytosis to produce an endosome, Domain T is the Translocation Domain which translocates the agent (with or without the Binding Site) from within the endosome across the endosomal membrane into the cytosol of the cell, Domain E is the Effector Domain which inhibits the ability of the Recyclable Membrane Vesicles to transport the Integral Membrane Proteins to the surface of the cell.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Continuation of U.S. application Ser. No.10/150,262, filed May 20, 2002, which is a Continuation of U.S.application Ser. No. 09/447,356, filed Nov. 22, 1999, which is aContinuation in part of U.S. application Ser. No. 08/945,037, filed Jan.12, 1998, which is a National Stage of U.S. Application PCT/GB96/00916,filed Apr. 16, 1996, based on U.K. application 9508204.6, filed Apr. 21,1995, incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Technical Field

This invention relates to a novel agent that is able to modifyperipheral afferent function. The agent may inhibit neurotransmitterrelease from discrete populations of neurons, and thereby reduce, orpreferably prevent, the transmission of afferent pain signals fromperipheral to central pain fibers. The agent may be used in or apharmaceutical for the treatment of pain, particularly chronic pain.

2. Background

The sense of touch has traditionally been regarded as one of the fiveclassical senses, but in reality it is highly complex, transducing anumber of different sensations. These sensations are detected in theperiphery by a variety of specialized nerve endings and associatedstructures. Some of these are specific for mechanical stimuli of varioussorts such as touch, pressure, vibration, and the deformation of hairsor whiskers. Another class of nerves is able to detect temperatures,with different fibers being activated by heat and cold. A furtherpopulation of nerve endings is not normally excited by mild stimuli, butby strong stimuli only. Sensory nerves of this category often respond tomore than one stimulus, and are known as high-threshold polymodalfibers. They may be used to sense potentially damaging situations orobjects. The polymodal fibers also transduce chemical signals such asthe “burning” sensation evoked by acid. Thus, the sense of touch cantransmit a very detailed description of objects and serve to both informand warn of events.

The transduction of sensory signals from the periphery to sensationitself is achieved by a multi-neuronal pathway and the informationprocessing centers of the brain. The first nerve cells of the pathwayinvolved in the transmission of sensory stimuli are called primarysensory afferents. The cell bodies for the primary sensory afferentsfrom the head and some of the internal organs reside in various of theganglia associated with the cranial nerves, particularly the trigeminalnuclei and the nucleus of the solitary tract. The cell bodies for theprimary sensory afferents for the remainder of the body lie in thedorsal root ganglia of the spinal column. The primary sensory afferentsand their processes have been classified histologically; the cell bodiesfall into two classes: A-type are large (60-120 μm in diameter) whileB-type are smaller (14-30 μm) and more numerous. Similarly the processesfall into two categories: C-fibers lack the myelin sheath that A-fiberspossess. A-fibers can be further sub-divided into Aβ-fibers, that arelarge diameter with well developed myelin, and Aδ-fibers, that arethinner with less well developed myelin. It is generally believed thatAβ-fibers arise from A-type cell both and that Aδ- and C-fibers arisefrom B-type cell bodies, These classifications can be further extendedand subdivided by studying the selective expression of a range ofmolecular markers.

Functional analyses indicate that under normal circumstances Aβ-fiberstransmit the senses of touch and moderate temperature discrimination,whereas the C-fibers are mainly equivalent to the polymodalhigh-threshold fibers mentioned above. The role of Aδ-fibers is lessclear as they seem to have a variety of responsive modes, with both highand low thresholds.

After the activation of the primary sensory afferents the next step inthe transduction of sensory signals is the activation of the projectionneurons, which carry the signal to higher parts of the central nervoussystem such as the thalamic nuclei. The cell bodies of these neurons(other than those related to the cranial nerves) are located in thedorsal horn of the spinal cord. This is also where the synapses betweenthe primary afferents and the projection neurons are located. The dorsalhorn is organized into a series of laminae that are stacked, with laminaI being most dorsal followed by lamina II, etc. The different classes ofprimary afferents snake synapses in different laminae. For cutaneousprimary afferents, C-fibers make synapses in laminae I and II, Aδ-fibersin laminae I, II, and V, and Aβ-fibers in laminae III, IV, and V. Deeperlaminae (V-VII, X) are thought to be involved in the sensory pathwaysarriving from deeper tissues such muscles and the viscera.

The predominant netter at the synapses between primary afferents andprojection neurons is glutamate, although importantly the C-fiberscontain several neuropeptides such as substance P and calcitonin-generelated peptide (CGRP). A-fibers may also express neuropeptides such asneuropeptide Y under some circumstances.

The efficiency of transmission of these synapses can be altered viadescending pathways and by local interneurons in the spinal cord. Thesemodulatory neurons release a number of mediators that are eitherinhibitory (e.g. opioid peptides, glycine) or excitatory (e.g. nitricoxide, cholecystokinin), to provide a mechanism for enhancing orreducing awareness of sensations.

A category of sensation that requires such physiological modulation ispain. Pain is a sensation that can warn of injury or illness, and assuch is essential in everyday life. There are times, however, when thereis a need to be able to ignore it, and physiologically this is afunction of, for example, the opioid peptides. Unfortunately, despitethese physiological mechanisms, pain can continue to be experiencedduring illnesses or after injuries long after its utility has passed. Inthese circumstances pain becomes a symptom of disease that would bebetter alleviated.

Clinically, pain can be divided into three categories: (1) Acute pain,usually arising from injury or surgery that is expected to disappearwhen that injury is healed. (2) Chronic pain arising from malignantdisease; the majority of people with metastatic cancer have moderate tosevere pain and this is resolved either by successful treatment of thedisease or by the death of the patient. (3) Chronic pain not caused bymalignant disease; this is a heterogeneous complaint, used by a varietyof illnesses, including arthritis and peripheral neuropathies, that areusually not life-threatening but which may last for decades withincreasing levels of pain.

The physiology of pain that results from tissue damage is betterunderstood than that which is caused by central nervous system defects.Under normal circumstances the sensations that lead to pain are firsttransduced by the Aδ- and C-fibers that carry high threshold signals.Thus the synapses in laminae I and II are involved in the transmissionof the pain signals, using glutamate and the peptides released byC-fibers to produce activation of the appropriate projection neurons.There is, however, evidence that in some chronic pain states otherA-fibers (including Aβ-fibers) can carry pain signals, and thus actprimary nociceptive afferents, for example in the hyperalgesia andallodynia associated with neuropathic pain. These changes have beenassociated with the expression of peptides such as neuropeptide Y in Afibers. During various chronic pain conditions the synapses of thevarious sensory afferents with projection neurons may be modified inseveral ways: there may be changes in morphology leading to an increasein the number of synapses, the levels and ratios of the differentpeptides may change, and the sensitivity of the projection neuron maychange.

Given the enormity of the clinical problem presented by pain,considerable effort has been expended in finding methods for itsalleviation. The most commonly used pharmaceuticals for the alleviationof pain fall into two categories: (1) Non-steroidal anti-inflammatorydrugs (NSAIDs), including aspirin and ibuprofen; (2) Opioids, includingmorphine.

NSAIDS have their main analgesic action at the periphery by inhibitingthe production of prostaglandins by damaged tissues. Prostaglandins havebeen shown to be peripheral mediators of pain and inflammation and areduction in their concentration provides relief to patients. This isespecially the case in mild arthritic disease, where inflammation is amajor cause of pain. It has been suggested that prostaglandins areinvolved in the mediation of pain in the spinal cord and the brain; thismay explain why NSAIDs have analgesic effects in some pain states thatdo not involve inflammation or peripheral tissue damage. Asprostaglandins, however, are only one of several mediators of painNSAIDs alone are only effective in reducing some types of mild pain toacceptable levels. They are regarded as having a ceiling of activityabove which increasing doses do not give increasing pain relief.Furthermore they have side effects that limit their usefulness inchronic complaints. The use of NSAIDs is associated with irritation ofthe gastro-intestinal tract and prolonged use may lead to thedevelopment of extensive ulceration of the gut. This is particularlytrue in elderly patients who form the largest cohort of patients with,for example, arthritis.

Opioids act at the level of the spinal cord to inhibit the efficiency ofneurotransmission between the primary nociceptive fibers (principallyC-fibers) and the projection neurons. They achieve this by causing aprolonged hyperpolarization of both elements of these synapses. The useof opioids is effective in alleviating most types of acute pain andchronic malignant pain. There are, however, a number of chronicmalignant pain conditions which are partly or completely refractory toopioid analgesia, particularly those which involve nerve compression,e.g. by tumor formation. Unfortunately opioids also have unwantedsystemic side-effects including: (1) depression of the respiratorysystem at the level of the respiratory centers in the brain stem; (2)the induction of constipation by a variety of effects on the smoothmusculature of the gastro-intestinal tract; and (3) psychoactive effectsincluding sedation and the induction of euphoria. These side effectsoccur at doses similar to those that produce analgesia and thereforelimit the doses that can be given to patients.

Delivery of opioids at the spinal level can reduce the side-effectprofile, but requires either frequently repeated spinal injections orfitting of a catheter, both of which carry increased risk to thepatient. Fitting of a catheter requires that the patient is essentiallyconfined to bed thus further restricting their quality of life.

The use of opioids for the treatment of some other types of chronic painis generally ineffective or undesirable. Examples include the painassociated with rheumatoid arthritis and neuromas that develop afternerve injury. The undesirable nature of opioid treatment in thesepatients is related not only to side-effects already mentioned and theprobable duration of the disease but also to the fourth majorside-effect of the opioids: dependence. Opioids such as morphine andheroin are well-known drugs of abuse that lead to physical dependence,this last side-effect involves the development of tolerance: the dose ofa drug required to produce the same analgesic effect increases withtime. This may lead to a condition in which the doses required toalleviate the pain are life-threatening due to the first threeside-effects.

Although NSAIDs and opioids have utility in the treatment of pain thereis general agreement that they are often not appropriate for theadequate treatment of pain, particularly chronic and severe pains.

Other treatments are also used, particularly for the treatment ofchronic severe pain including surgical lesions of the pain pathways atseveral levels from peripheral nerves through dorsal root section andcordotomy to pituitary destruction. These are, however, mostly severeoperations that are all associated with significant risk to the patient.

It can be seen, therefore, that there remains a significant need for thedevelopment of new classes of pharmaceuticals for the treatment of painof many types. The desired properties of such new therapies can bebriefly expressed as follows: (1) the ability to provide significantrelief of pain including severe pain; (2) the lack of systemic sideeffects that significantly impair the patients quality of life; (3)long-lasting actions that do not require frequent injections orlong-term catheterization of patients; (4) provision of agents that donot lead to tolerance and associated dependence.

STATEMENT OF INVENTION

The present invention relates to an agent which can reduce andpreferably prevent the transmission of pain signals from the peripheryto the central nervous system, thereby alleviating the sensation ofpain. Specifically, the invention can provide an agent which can reduceand preferably prevent the transmission of pain signals from nociceptiveafferents to projection neurons. More specifically, the invention canprovide an agent which can inhibit the exocytosis of at least oneneurotransmitter or neuromodulator substance from at least one categoryof nociceptive afferents.

In a first aspect of the invention, there is provided an agent which canbe administered systemically, and can specifically target definedpopulations of nociceptive afferents to inhibit the release of at leastone neurotransmitter or neuromodulator from the synaptic terminals ofnerves.

In a second aspect of the invention, there is provided an agent whichcan be locally administered at the periphery, and which is able toinhibit the release of at least one neurotransmitter or neuromodulatorfrom the synaptic terminals of nociceptive afferents transmitting thepain signal from the periphery.

In a third aspect of the invention, an agent is provided which can beadministered into the spinal cord, and which can inhibit the release ofat least one neurotransmitter or neuromodulator from the synapticterminals of nociceptive afferents terminating in that region of thespinal cord.

In a fourth aspect of the invention, there is provided an agent whichcan specifically target defined populations of afferent neurons, so thatthe effect of the agent is limited to that cell type.

In a fifth aspect of the invention, there is provided a method oftreatment of pain which comprises administering an effective dose of theagent according to the invention.

In a sixth aspect of the invention, the agent can be expressedrecombinantly as a fusion protein which includes the required componentsof the agent

DEFINITIONS

Without wishing to be limited by the definitions set down, it isintended in this description that the following terms have the followingmeanings:

Light chain means the smaller of the two polypeptide chains which formclostridial neurotoxins; it has a molecular mass of approximately 50 kDaand is commonly referred to as L-chain or simply L.

Heavy chain means the larger of the two polypeptide chains which formclostridial neurotoxins; it has a molecular mass of approximately 100kDa and is commonly referred to as H-chain or simply H.

H_(C) fragment means a fragment derived from the H-chain of aclostridial neurotoxin approximately equivalent to the carboxy-terminalhalf of the H-chain, or the domain corresponding to that fragment in theintact H-chain. It contains the domain of the natural toxin involved inbinding to motor neurons.

H_(N) fragment means a fragment derived from the H-chain of aclostridial neurotoxin approximately equivalent to the amino-terminalhalf of the H-chain, or the domain corresponding to that fragment in theintact in the H-chain. It contains a domain involved in thetranslocation of the L-chain across endosomal membranes.

LH_(N) means a fragment derived from a clostridial neurotoxin thatcontains the L-chain, or a functional fragment thereof coupled to theH_(N) fragment. It is commonly derived from the intact neurotoxin byproteolysis.

Targeting Moiety (TM) means any chemical structure of an agent whichfunctionally interacts with a binding site causing a physicalassociation between the agent and the surface of a primary sensoryafferent.

Binding site (BS) means a structure on the surface of a cell with whichexogenous molecules are able to interact in such a way as to bring abouta physical association with the cell.

Primary sensory afferent is a nerve cell that can carry sensoryinformation from the periphery towards the central nervous system.

Primary nociceptive afferent is a nerve cell that can carry sensoryinformation from the periphery towards the central nervous system, wherethat information can result in a sensation of pain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a Coomassie stain of an SDS-PAGE analysis of the fractionsfrom size-exclusion chromatography of the products of the couplingreaction between derivatized Nerve Growth Factor (NGF) and derivatizedLH_(N) from BoNT/A.

FIG. 2 shows a Coomassie stain of an SDS-PAGE analysis of the conjugateof NGF and LH_(N) under reducing and non-reducing conditions.

FIG. 3 shows a Western blot of extracts from PC12 cells treated with theconjugate of NGF and LH_(N), probed with an antibody that recognizes theproduct of the proteolysis of SNAP-25 by the L-chain of BoNT/A.

FIG. 4 shows a Western blot of extracts from rat dorsal root ganglionneurons treated with the conjugate of NGF and LH_(N), probed with anantibody that recognizes the product of the proteolysis of SNAP-25 bythe L-chain of BoNT/A.

DETAILED DESCRIPTION OF THE INVENTION

It can be seen that, an agent for reducing or preventing thetransmission of pain signals from peripheral, nociceptive afferentneurons to projection neurons has many potential applications in thereduction of the sensation of pain, particularly of severe, chronicpain.

According to the invention, there is provided an agent which can inhibitthe release of at least one neurotransmitter or neuromodulator or bothfrom the synaptic terminals of nociceptive afferents.

The agent has a number of discrete functions:

-   1) It binds to a surface structure (the Binding Site [BS]) which is    characteristic of, and has a degree of specificity for, nociceptive    afferent neurons.-   2) It enters the neuron. The entry of molecules into a cell can    occur by a process of endocytosis. Only certain cell surface BSs    undergo endocytosis, and preferably the BS to which the agent binds    is one of these. In one aspect of this invention, the BS is present    on the peripheral, sensory fibers of the nociceptive afferent neuron    and, following internalization, undergoes retrograde transport to    the cell body and central processes of the neuron, in such a manner    that the agent is also delivered to these regions of the neuron. In    another aspect of this invention, the BS to which the agent binds is    present on the central processes or cell body of the nociceptive    afferent neuron.-   3) The agent enters the cytosol.-   4) The agent modifies components of the exocytotic machinery present    in the synaptic terminals of the central processes of those neurons,    such that the release of at least one neurotransmitter or    neuromodulator from the synaptic terminal is reduced or preferably    prevented.

Surprisingly, an agent of the present invention can be produced bymodifying a clostridial neurotoxin or fragment thereof.

The clostridial neurotoxins are proteins with molecular masses of theorder of 150 kDa. They are produced by various species of the genusClostridium, most importantly C. tetani and several stains of C.botulinum. There are at present eight different classes of theneurotoxins known: tetanus toxin, and botulinum neurotoxin in itsserotypes A, B, C1, D, E, F and G, and they all share similar structuresand modes of action. The clostridial neurotoxins are synthesized by thebacterium as a single polypeptide that is modified post-translationallyto form two polypeptide chains joined together by a disulphide bond. Thetwo chains are termed the heavy chain (H), which has a molecular mass ofapproximately 100 kDa, and the light chain (L), which has a molecularmass of approximately 50 kDa. The clostridial neurotoxins bind to anacceptor site on the cell membrane of the motor neuron at theneuromuscular junction and are internalized by an endocytotic mechanism.The internalized clostridial neurotoxins possess a highly specificzinc-dependent endopeptidase activity that hydrolyses a specific peptidebond in at least one of three proteins, synaptobrevin, syntaxin orSNAP-25, which are crucial components of the neurosecretory machinery,and this activity of the clostridial toxins results in a prolongedmuscular paralysis. The zinc-dependent endopeptidase activity ofclostridial neurotoxins is found to reside in the L-chain. Theclostridial neurotoxins are highly selective for motorneurons due to thespecific nature of the acceptor site on those neurons. The specificneuromuscular junction binding activity of clostridial neurotoxins isknown to reside in the carboxy-terminal portion of the heavy chaincomponent of the dichain neurotoxin molecule, a region known as H_(C).

Surprisingly, by covalently linking a clostridial neurotoxin, or ahybrid of two clostridial neurotoxins, in which the H_(C) region of theH-chain has been removed or modified, to a new molecule or moiety, theTargeting Moiety (TM), that binds to a BS on the surface of sensoryneurons, a novel agent capable of inhibiting the release of at least oneneurotransmitter or neuromodulator from nociceptive afferents isproduced. A further surprising aspect of the present invention is thatif the L-chain of a clostridial neurotoxin, or a fragment of the L-chaincontaining the endopeptidase activity, is covalently linked to a TMwhich can also effect internalization of the L-chain, or fragmentthereof, into the cytoplasm of a sensory neuron, this also produces anovel agent capable of inhibiting the release of at least oneneurotransmitter or neuromodulator. The covalent linkages used to couplethe component parts of the agent may include appropriate spacer regions.

The TM provides specificity for the BS on the nociceptive afferentneuron. The TM component of the agent can comprise one of many cellbinding molecules, including, but not limited to, antibodies, monoclonalantibodies, antibody fragments (Fab, F(ab)′2, Fv, ScFv, etc.), lectinsand ligands to the receptors for hormones, cytokines, growth factors orneuropeptides. A list of possible TMs is given in Table 1, this list isillustrative and is not intended to be limiting to the scope of TMswhich could fulfill the requirements of this invention. In oneembodiment of the invention the TM binds to a BS which undergoesretrograde transport.

It is known in the art that the H_(C) portion of the neurotoxin moleculecan be removed from the other portion of the heavy chain, known asH_(N), such that the H_(N) fragment remains disulphide linked to thelight chain (L-chain) of the neurotoxin molecule to provide a fragmentknown as LH_(N). Thus, in one embodiment of the present invention theLHN fragment of a clostridial neurotoxin is covalently linked, usinglinkages which may include one or more spacer regions, to a TM.

In another embodiment of the invention, the H_(C) domain of aclostridial neurotoxin is mutated or modified, e.g. by chemicalmodification, to reduce or preferably incapacitate its ability to bindthe neurotoxin to receptors at the neuromuscular Junction. This modifiedclostridial neurotoxin is then covalently linked, using linkages whichmay include one or more spacer regions, to a TM.

In another embodiment of the invention, the heavy chain of a clostridialneurotoxin, in which the H_(C) domain is mutated or modified, e.g. bychemical modification, to reduce or preferably incapacitate its abilityto bind the neurotoxin to receptors at the neuromuscular junction iscombined with the L-chain of a different clostridial neurotoxin. Thehybrid, modified clostridial neurotoxin is then covalently linked, usinglinkages which may include one or more spacer regions, to a TM.

In another embodiment of the invention, the H_(N) portion of aclostridial neurotoxin is combined with the L-chain of a differentclostridial neurotoxin. The hybrid LH_(N) is then covalently linked,using linkages which may include one or more spacer regions, to a TM.

In another embodiment of the invention the light chain of a clostridialneurotoxin, or a fragment of the light chain containing theendopeptidase activity, is linked, using linkages which may include oneor more spacer regions, to a TM which can also effect theinternalization of the light chain, or fragment thereof containingendopeptidase activity, into the cytoplasm of the cell.

In another embodiment of the invention the agent is expressedrecombinantly as a fusion protein which includes an appropriate fragmentof a Targeting Moiety in addition to any desired spacer domains. Therecombinantly expressed agent may be derived wholly from the geneencoding one serotype of neurotoxin or be a chimaera derived from thegenes encoding two different serotypes.

In this embodiment, a genetic construct is employed which encodes theclostridial neurotoxin, or fragment thereof, and the TM. Reference tothe clostridial neurotoxin includes reference to a fragment thereofhaving the desired protease activity.

The coding sequences of the TM and clostridial neurotoxin are preferablyarranged in a single genetic construct. These coding sequences arepreferably arranged in-frame so that subsequenttranscription/translation is continuous through both coding sequencesand results in a fusion protein.

Alternatively, the coding sequences of the TM and clostridial neurotoxinmay be arranged on separate genetic constructs and, followingtranslation, the corresponding proteins associate with each other toform the agent. Association of the TM and clostridial neurotoxintranslation products may be encouraged by ensuring that each translationproduct has one or more mutually compatible amino acids at an exposedsurface. An example of such an amino acid is cysteine, or othersulphur-containing amino acids. The presence of a sulphur group on theseamino acids allows the formation of disulphide bridges between the TMand clostridial neurotoxin translation products.

The fusion protein aspect of the present invention may employ anyvariation of any TM-LH_(N) sequence identified in the presentspecification. For example:

-   -   N and C-terminal protein fusions (ie. either 5′ or 3′ genetic        construct fusions) may be employed. Different combinations of        TM-LH_(N), LH_(N)-TM and L-TM-H_(N) may be more suitable than        others;    -   should a L-TM-H_(N) fusion be employed then it may be preferable        to insert a specific cleavage sequence between the L-chain and        TM to enable the TM to have freedom to bind to the target        receptor. According to this embodiment it may be preferable to        engineer into the genetic construct means (eg. a disulphide        bridge) to keep the fusion complex together;        the genetic construct preferably incorporates a nucleic acid        sequence encoding a spacer peptide at the TM/LH_(N) fusion        junction. However, a spacer is not essential. Examples of spacer        peptides include:    -   PPPIEGR [Kim, J. S., Raines, R. T. (1993). Ribonuclease        S-peptide as a carrier in fusion proteins, Protein Sci        2(3):348-56];    -   collagen-like spacer (Rock, F., Everett, M., Klein, M. (1992).        Over-expression and structure-function analysis of a        bioengineered IL-2/IL-6 chimeric lymphokine. Protein Eng        5(6):583-91]; and    -   trypsin sensitive diphtheria toxin peptide (O'Hare, M.,        Brown, A. N., Hussain, K., Gebhardt, A., Watson, G., Roberts, L.        M., Vitetta, E. S., Thorpe, P. E., Lord, J. M. (1990).        Cytotoxicity of a recombinant ricin-A-chain fusion protein        containing a proteolytically-cleavable spacer sequence. FEBS        Lett October 29;273(1-2):200-4].

Turning to the clostridial neurotoxin component of the agent, all LH_(N)variants described in the present application and in the presentApplicant's co-pending application U.S. Ser. No. 09/255,829 may beemployed. The content of U.S. Ser. No. 09/255,829 is herein incorporatedby reference thereto.

All constructs have a 5′ ATG codon to encode an N-terminal methionineand a C-terminal translational stop codon if these codons are notalready present. Expression of a number of fusion proteins is well-knownin the art and was so at the priority date of the present application(ie. Apr. 21, 1995). Methods for the construction and expression of theconstructs of the present invention may employ information from thefollowing references and others:

-   Lorberboum-Galaki, H., Fitzgerald, D., Chaudhary, V., Adhya, S.,    Pastan, I. (1988). Cytotoxic activity of en Interleukin    2-Pseudomonas exotoxin chimeric protein produced in Escherichia    coli. Proc Natl Aced Sci USA 85(6):1922-6;-   Murphy, J. R. (1988) Diphtheria-related peptide hormone gene    fusions: a molecular genetic-approach to chimeric toxin development.    Cancer Treat Res; 37:123-40;-   Williams, D. P., Parker, K., Bacha, P., Bishal, W., Borowski, M.,    Genbauffe, F., Strom, T. B., Murphy, J. R. (1987). Diphtheria toxin    receptor binding domain substitution with interleukin-2: genetic    construction and properties of a diphtheria toxin-related    interleukin-2 fusion protein. Protein Eng; 1(6):493-8;-   Arora, N., Williamson, L. C., Leppla, S. H., Halpern, J. L. (1994).    Cytotoxic effects of a chimeric protein consisting of tetanus toxin    light chain and anthrax toxin lethal factor in non-neuronal cells J    Biol Chem; 269(42):26165-71;-   Brinkmann, U., Reiter, Y., Jung, S. H., Lee, B., Pastan, I. (1993).    A recombinant Immunotoxin containing a disulphide-stabilized Fv    fragment. Proc Natl Acad Sci USA; 90(16):7538-42; and-   O'Hare, M., Brown, A. N., Hussain, K., Gebhardt, A., Watson, G.,    Roberts, L. M., Vitetta, E. S., Thorpe, P. E., Lord, J. M. (1990).    Cytotoxicity of a recombinant ricin-A-chain fusion protein    containing a proteolytically/-cleavable spacer sequence. FEBS Left    October 29;273(1-2):200-4.

The method of preparing a fusion protein according to the presentinvention requires nucleic acid sequence data relating to the selectedTM and the clostridial neurotoxin. These sequence data were readilyavailable at the priority date of the present application as evidencedby the data/publications of several preferred TMs which have been listedin the present specification. Alternatively, any necessary sequence datamay he obtained by techniques well-known to the skilled person.

In one embodiment, DNA encoding the TM sequences may be cloned from asource organism by screening a cDNA library for the correct codingregion (for example by using specific oligonucleotides based on theknown sequence information to probe the library), isolating the TM DNA,sequencing this DNA for confirmation purposes, and then placing theisolated DNA in an appropriate expression vector for expression in thechosen host.

As an alternative to isolation of the sequence from a library, theavailable sequence information may be employed to prepare specificprimers for use in PCR, whereby the coding sequence is then amplifieddirectly from the source material and, by suitable use of primers, maybe cloned directly into an expression vector.

Another alternative method for isolation of the coding sequence is touse the existing sequence information and synthesize a copy, possiblyincorporating alterations, using DNA synthesis technology.

Another alternative method is to use existing protein sequenceinformation and synthesize a version of the coding sequence that cangive rise to that protein sequence. Using DNA synthesis technology to dothis (and the alternative described above) enables the codon bias of thecoding sequence to be modified to be optimal for the chosen expressionhost. This may give rise to superior expression levels of the fusionprotein.

All of the above methods may be employed to obtain sequence informationon the selected clostridial neurotoxin component of the agent.

Ideally, optimization of the codon bias for the expression host would beapplied to the TM, the spacer (if there is one), and the LH_(N) variant.Optimization of the codon bias is possible by application of the proteinsequence into freely available DNA/protein database software, eg.programs available from Genetics Computer Group, Inc.

By way of example, the following TM sequences were readily available bythe priority date of the present application (ie. Apr. 21, 1995).

1) Human B-NGF Precursor

-   -   ULLRICH, A., GRAY, A., BERMAN, C., DULL, T. J. (1983). “Human        beta-nerve growth factor gene sequence highly homologous to that        of mouse.” Nature 303:821-825.    -   BORSANI, G., PIZZUTI, A., RUGARLI, E. I., FALINI, A.,        SILANI, V. R. A., SIDOLI, A., SCARLATO, G., BARELLE, F. E.        (1990). “CDNA sequence of human beta-NGF.” Nucleic Acids Res.        18:4020-4020.        Sequence data:

Mouse beta-nerve growth factor (beta-NGF) MRNA. [ACCESSION K01759] H.sapiens gene for beta-nerve growth factor (beta-NGF). [ACCESSION V01511](SEQ ID NO: 1) Mature NGF sequence = SSSHPIFHRG EFSVCDSVSV WVGDKTTATDIKGKEVMVLG EVNINNSVFK 51 QYFFETKCRD PNPVDSGCRG IDSKHWNSYC TTTHTFVKALTMDGKQAAWR 101 FIRIDTACVC VLSRKAVRRA

Back-translated bacterial codon usage sequence for NGF. This, and all ofthe following sequences, are RNA sequences, and for cloning purposeswould need to be the DNA version with T instead of U (SEQ ID NO:2):UCCUCCUCCC ACCCGAUCUU CCACCGUGGU GAAUUCUCCG UUUGCGACUC 51 CGUUUCCGUUUGGGUUGGUG ACAAAACCAC CGCUACCGAC AUCAAAGGUA 101 AAGAAGUUAU GGUUCUGGGUGAAGUUAACA UCAACAACUC CGUUUUCAAA 151 CAGUACUUCU UCGAAACCAA AUGCCGUGACCCGAACCCGG UUGACUCCGG 201 UUGCCGUGOU AUCGACUCCA AACACUGGAA CUCCUACUGCACCACCACCC 251 ACACCUUCGU UAAAGCUCUG ACCAUGGACG GUAAACAGGC UGCUUGGCGU301 UUCAUCCGUA UCGACACCGC UUGCGUUUGC GUUCUGUCCC GUAAAGCUGU 351UCGUCGUGCU

The following sequence is the precursor for NGF. This pro-sequence maybe useful in certain expression situations. However the mature sequencegiven above would be preferred. (SEQ ID NO:3) Pro-NGF sequence =MSMLFYTLIT AFLIGIQAEP HSESNVPAGH TIPQVHWTKL QHSLDTALRR 51 ARSAPAAAIAARVAGQTRNI TVDPRLFKKR RLRSPRVLFS TQPPREAADT 101 QDLDFEVGGA APFNRTHRSKRSSSHPIFHR GEFSVCDSVS VWVGDKTTAT 151 DIKGKEVMVL GEVNINNSVF KQYFFETKCRDPNPVDSGCR GIDSKKWNSY 201 CTTTHTFVKA ITMDGKQAAW RFIRIDTACV CVLSRKAVRR A

Back-translated bacterial codon usage-sequence for pro-NGF (SEQ IDNO:4)= AUGUCCAUGC UGUUCUACAC CCUGAUCACC GCUUUCCUGA UCGGUAUCCA 51GGCUGAACCG CACUCCGAAU CCAACGUUCC GGCUGGUCAC ACCAUCCCGC 101 AGGUUCACUGGACCAAACUG CAGCACUCCC UGGACACCGC UCUGCGUCGU 151 GCUCGUUCCG CUCCGGCUGCUGCUAUCGCU GCUCGUGUUG CUGGUCAGAC 201 CCGUAACAUC ACCGUUGACC CGCGUCUGUUCAAAAAACGU CGUCUGCGUU 261 CCCCGCGUOU UCUAUUCUCC ACCCAGCCGC CGCGUGAAGCUGCUGACACC 301 CAGGACCUGG ACUUCGAAGU UGGUGGUGCU GCUCCGUUCA ACCGUACCCA361 CCGUUCCAAA CGUUCCUCCU CCCACCCGAU CUUCCACCGU GGUGAAUUCU 401CCGUUUGCGA CUCCGUUUCC GUUUGGGUUG GUGACAAAAC CACCGCUACC 451 GACAUCAAAGGUAAAGAAGU UAUGGUUCUG GGUGAAGUUA ACAUCAACAA 501 CUCCGUUUUC AAACAGUACUUCUUCGAAAC CAAAUGCCGU GACCCGAACC 561 CGGUUGACUC CGGUUGCCGU GGUAUCUACUCCAAACACUG GAACUCCUAC 801 UGCACCACCA CCCACACCUU CGUUAAAGCU CUGACCAUGGACGGUAAACA 651 GGCUGCUUGG CGUUUCAUCC GUAUCGACAC CGCUUGCGUU UGCGUUCUGU701 CCCGUAAAGC UGUUCGUCGU GCU2) Enkephalin:

-   -   NODA, M., TERANISHI, Y., TAKAHASHI, H., TOYOSATO, M., NOTAKE,        M., NAKANISHI, S., NUMA, S. (1982). “Isolation and structural        organization of the human preproenkephalin gene.”; Nature        297:431-434.    -   COMB, M., SEEBURG, P. H., ADELMAN, J., EIDEN, L., HERBERT, E.        (1982). “Primary structure of the human Met- and Leu-enkephalin        precursor and its MRNA.”; Nature 295:663-666.        Sequence data:

Leu-enkephalin=YGGFL (SEQ ID NO:5) therefore a corresponding bacterialcoding sequence would be (SEQ ID NO:6) 1 UACGGUGGUU UCCUG

Met-enkephalin=YGGFM (SEQ ID NO:7) therefore a corresponding bacterial15 coding sequence would be (SEQ ID NO:8) 1 UACGGUGGUU UCAUG3) Bradykinin:

-   -   TAKAGAKI, Y., KITAMURA, N., NAKANISHI, S. (1985). “Cloning and        sequence analysis of cDNAs for human high molecular weight end        low molecular weight prekininogens. Primary structures of two        human prekininogens.”; J. Biol. Chem. 260:8601-8609.        Sequence data:

Protein sequence=RPPGFSPFR (SEQ ID NO:9) Therefore a correspondingbacterial coding sequence would be (SEQ ID NO:10)

CGUCCGCCGG GUUUCUCCCC GUUCCGU

In another embodiment of the invention the required LH_(N), which may bea hybrid of an L and H_(N) from different clostridial toxin types, isexpressed recombinantly as a fusion protein with the TM, and may alsoinclude one or more spacer regions.

In another embodiment of the invention the light chain of a clostridialneurotoxin, or a fragment of the light chain containing theendopeptidase activity, is expressed recombinantly as a fusion proteinwith a TM which can also affect the internalization of the light chain,or fragment thereof containing the endopeptidase activity, into thecytoplasm of the cell. The expressed fusion protein may also include oneor more spacer regions.

The basis of this disclosure is the creation of novel agents with veryspecific and defined activities against a limited and defined class ofneurons (primary sensory afferents), and as such the agents may beconsidered to represent a form of neurotoxin. The therapeutic use ofnative botulinum neurotoxins is well known in the prior art. The mode ofaction of the botulinum neurotoxins, as described in the prior art,however, is by a mechanism, inhibition of acetylcholine secretion, andagainst a category of target neurons, efferent motorneurons, clearlydistinct from the agents described in this disclosure. The prior artdoes not teach either the activity or the chemical structure of theagents disclosed. Thus, although, as discussed in this application, theprior art teaches much about the native clostridial neurotoxins, nativeunmodified clostridial neurotoxins are not the subject of thisdisclosure. The agent of this invention requires modification of theclostridial neurotoxins such that the targeting property taught in theprior art is removed. The modified neurotoxin is then coupled to a newtargeting function (the TM), to give a novel agent with new biologicalproperties distinct from those of the native clostridial neurotoxins andnot taught in the prior art. It is this new agent with novel propertiesthat is the subject of this disclosure.

Exploitation in Industry

The agent described in this invention can be used in vivo, eitherdirectly or as a pharmaceutically acceptable salt, for treatment ofpain.

For example, an agent according to the invention can be usedsystemically for the treatment of severe chronic pain. A specificexample of this is the use in treatment of clinical pain associated withrheumatoid arthritis affecting multiple joints.

In another example, an agent according to the invention can be locallyapplied for the treatment of pain. A specific example of this istreatment by local injection into a joint affected by inflammatory pain.

In further example an agent according to the invention can beadministered by spinal injection (epidural or intrathecal) at the levelof the spinal segment involved in the innervation of an affected organfor the treatment of pain. This is, for example, applicable in thetreatment of deep tissue pain, such as chronic malignant pain.

The present invention will now be illustrated by reference to thefollowing non-limiting examples:

EXAMPLE 1 Synthesis of a Conjugate of NGF and the LH_(N) Fragment ofBoNT/A

Lyophilised murine 2.5 S NGF was dissolved by the addition of water anddialysed into MES buffer (0.1 M MES, 0.1 M sodium chloride, pH 5.0). Tothis solution (at a concentration of about 0.3 mg/ml) was added PDPH(100 mg/ml in DMF)to a final concentration of 1 mg/ml. After mixing,solid EDAC was added to produce a final concentration of about 0.2mg/ml. The reaction was allowed to proceed for at least 30 min at roomtemperature. Excess PDPH was then removed by desalting over a PD-10column (Pharmacia) previously equilibrated with MES buffer.

The LH_(N) fragment of BoNT/A was produced essentially by the method ofShone C. C., Hambleton, P., and Melling, J. 1987, Eur. J. Biochem. 167,175-180. An amount of LH_(N) equivalent to half the weight of NGF useddissolved in triethanolamine buffer (0.02 M triethanolamine/HCl, 0.1 Msodium chloride, pH 7.8) at a concentration of about 1 mg/ml, wasreacted with Traut's reagent (100 mM stock solution in 1 Mtriethanolamine/HCl, pH 8.0) at a final concentration of 2 mM. After onehour the LH_(N) was desalted into PBSE (phosphate buffered saline with 1mM EDTA) using a PD-10 column (Pharmacia). The protein peak from thecolumn eluate was concentrated using a Microcon 50 (Amicon) to aconcentration of about 2 mg/ml.

The derivatized NGF was subjected to a final concentration stepresulting in a reduction in volume to less than 10% of the startingvolume and then mixed with the derivatized LH_(N) overnight at momtemperature. The products of the reaction were analysed bypolyacrylamide gel electrophoresis in the presence of sodiumdodecyl-sulphate (SDS-PAGE).

The conjugate resulting from the above reaction was partially purifiedby size exclusion chromatography over Bio-Gel P-100 (Bio-Rad). Theelution profile was followed by measuring the optical density at 280 nmand SDS-PAGE analysis of the fractions. This allowed the separation ofconjugate from free NGF and by-products of the reaction.

FIG. 1 shows the SDS-PAGE analysis of the fractions from one suchBio-Gel P-100 column. The free LH_(N) and conjugate (M_(r) 100 kDa andabove) are clearly separated from the majority of the free NGF (M_(r) 13kDa). As 2.5 S NGF is a homo-dimer formed by non-covalent interactionsit is dissociated by treatment with SDS. Thus molecules that have formedcovalent cross-links to LH_(N) through one subunit only will dissociateduring the SDS-PAGE analysis and give rise to the free NGF band seen infractions 4-6. This result demonstrates that the homo-dimeric structureof NGF remains intact after derivatisation. The free LH_(N) seen inthese fractions represents a minor component which has not coupled toNGF. Fractions 4-6 were pooled before thither analysis.

FIG. 2 shows an analysis of the conjugate by SDS-PAGE under reducing andnon-reducing conditions. Lane 1 is free LH_(N) under non-reducingconditions, lane 2 is the same amount of LH_(N) reduced with 50 mMdithiothreitol. Lanes 3 and 4 show the conjugate after size exclusionchromatography either without (lane 3) or with (lane 4) reduction bydithiothreitol. Similarly, lanes 5 and 6 show NGF without or withreduction respectively. The results clearly show that the material inlane 5 with an apparent molecular mass greater than 100 kDa produces,upon reduction, the constituent bands of LH_(N) and NGF only.Furthermore the intensity of the bands following reduction is such thatthey must be derived from material other than the small amounts of freeLH_(N) and NGF observed in the unreduced sample. The only availablesource for the excess is the material with an apparent molecularmass>100 kDa. The conjugate in the fractions obtained following thesize-exclusion chromatography thus represents NGF and LH_(N) covalentlylinked by reducible disulphide linkages.

The fractions containing conjugate were stored at 4° C. until required.

EXAMPLE 2 Activities of a Conjugate of NGF and LH_(N) in PC-12 Cells

PC12 cells are a cell-line of neuroectodermal derivation that arecommonly used as a model system for the study of nerve function. As amodel system for testing the function of a conjugate of NGF and LH_(N)they have two necessary features: firstly they are well known to possesscell-surface receptors for NGF that have been shown to be involved in adifferentiation process in response to low concentrations of NGF.Secondly they have been shown to contain the exocytotic machinery forneurotransmitter release including, importantly in this example,SNAP-25.

PC12 cells were plated out into a 24-well plate that had been coatedwith MATRIGEL basement membrane matrix (Collaborative BiomedicalProducts) at a density of approximately 5×10⁵ cells per well. After afew days in culture (RPMI 1640 with 2 mM glutamine, 10% horse serum and5% foetal calf serum, 37° C., 5% CO₂) the medium was replaced with freshmedium containing added conjugate (prepared as described in Example 1)or LH_(N) or no addition. After being kept in culture overnight themedium was removed and the cells washed once with fresh medium. Cellswere then lysed by the addition of 0.45 ml sodium hydroxide (0.2 M) for30 min. After this time the solutions were neutralized by the additionof 0.45 ml hydrochloric acid (0.2 M) followed by 0.1 ml of HEPES/NaOH (1M, pH 7.4). To extract the membrane proteins from these mixturesTriton-X-114 (10%, v/v) was added and incubated at 4° C. for 60 min, theinsoluble material was removed by centrifugation and the supernatantswere then warmed to 37° C. for 30 min. The resulting two phases wereseparated by centrifugation and the upper phase discarded. The proteinsin the lower phase were precipitated with chloroform/methanol foranalysis by Western blotting.

The samples were separated by SDS-PAGE and transferred tonitro-cellulose. Proteolysis of SNAP-25, a crucial component of theneurosecretory process and the substrate for the zinc-dependentendopeptidase activity of BoNT/A, was then detected by probing with anantibody that recognizes the newly revealed carboxy-terminus of thecleaved SNAP-25 (the antibody is described in Patent ApplicationPCT/GB95/01279). FIG. 3 shows an example of such a Western blot. Nosignificant immunoreactivity was observed in samples from control cells(lanes 1 and 2) whereas a band corresponding to a molecular mass of 29kDa was observed weakly in samples incubated with 10 mg/ml LH_(N) (lanes5 and 6) and strongly in samples incubated with 10 mg/ml of theconjugate of NGF and LH_(N) (lanes 3 and 4). Thus incubation of PC12cells with the conjugate leads to the marked proteolysis of SNAP-25indicting that the conjugate has introduced the zinc-dependentproteolytic activity of the L-chain of BoNT/A into the cells' cytoplasm.Little or no such activity was seen with the constituent components ofthe conjugate.

Incubation of cells with the conjugate in the presence of an excess offree NGF resulted in a reduced production of the proteolytic product ofSNAP-25 than did incubation with the conjugate alone. This indicatesthat the action of the conjugate occurs by means of the NGF targetingmoiety interacting with the cell surface receptors for NGF.

EXAMPLE 3 The Activity of a Conjugate of NGF and LH_(N) in PrimaryCultures of Dorsal Root Ganglion Neurons

The dorsal root ganglia contain the cell bodies of primary nociceptiveafferents. It is well established that in primary in vitro cultures ofthis tissue the neurons retain many of the characteristics of thenociceptive afferents. These characteristics include the ability torelease neuropeptides such as substance P in response to chemicalstimuli known to cause pain in vivo (e.g. capsaicin). Furthermore theneurons are known to possess receptors for NGF.

Primary cultures of dorsal root ganglion neurons were establishedfollowing dissociation of the ganglia dissected from rat embryos(embryological age 12-15 days). The cells were plated into 12 wellplates at an initial density of 3×10⁵ cells/well in a medium containingNGF (100 ng/ml). After one day in culture fresh medium containingcytosine arabinoside (10 mM) was added to kill non-neuronal cells. Thecytosine arabinoside was removed after 2-4 days. Alter several more daysin culture the medium was replaced with fresh medium containingconjugate or LH_(N) in the absence of NGF. Following overnightincubation at 37° C. the medium was removed, the cells were lysed andthe hydrophobic proteins extracted using Triton-X-114 as described inExample 2.

The samples were analyzed by Western blotting as described in Example 2with the antibody that recognizes the product of the BoNT/A proteolysisof SNAP-25. No immunoreactivity was observed in samples from controlcells (lane 4) whereas a band corresponding to a molecular mass of 29kDa was observed weakly in samples incubated with 10 mg/ml LH_(N) (lane3) and strongly in samples incubated with 10 mg/ml of the conjugate ofNGF and LH_(N) (lanes 1 and 2).

This result indicates that the conjugate can deliver theproteolytically-active L-chain of BoNT/A into the cytoplasm of theneuronal cells that, in vivo, form the primary nociceptive afferents.

EXAMPLE 4 The Production of a Chimeric LH_(N) whereof the L Chain isDerived from BoNT/B and the H_(N) Fragment from BoNT/A

The H_(N) fragment of BoNT/A is produced according to the methoddescribed by Shone C. C., Hambleton, P., and Melling, J. (1987, Eur. J.Biochem. 167, 175-180) and the L-chain of BoNT/B according to the methodof Sathyamoorthy, V. and DasGupta, B. R. (1985, J. Biol. Chem. 260,10461-10466). The free cysteine on the H_(N) fragment of BoNT/A is thenderivatised by the addition of a ten-fold molar excess of dipyridyldisulphide followed by incubation at 4° C. overnight. The excessdipyridyl disulphide and the thiopyridone by product are then removed bydesalting the protein over a PD10 column (Pharmacia) into PBS.

The derivatised H_(N) is then concentrated to a protein concentration inexcess of 1 mg/ml before being mixed with an equimolar portion ofL-chain from BoNT/B (>1 mg/ml in PBS). After overnight incubation atroom temperature the mixture is separated by size exclusionchromatography over Superose 6 (Pharmacia), and the fractions analyzedby SDS-PAGE. The chimeric LH_(N) is then available for derivatisation toproduce a targeted conjugate as described in Example I.

EXAMPLE 5 A Method for Expression of a Protein Fusion Construct

The following references relate to the expression of recombinant NGF:

-   -   Dicou, E. (1992) Expression of recombinant human nerve growth        factor in Escherichia coli, Neurochem Int. 20, 129-134; and    -   Fujimori, K et al. (1992) Overproduction of biologically-active        human nerve growth factor in Escherichia coli,        Biosci-Biotechnol-Biochem, 56, 1985-1990.

This example describes how to make a NGF-LH_(N)/A fusion construct.

The coding region far mature NGF is placed in frame with the codingsequence for LH_(N)/A such that translation of protein is continuousthrough the NGF and into LH_(N)/A coding sequence.

Translation is initiated by incorporation of the codon for an N-terminalmethionine (ATG) immediately before the NGF sequence.

Translation is terminated by incorporation of a STOP codon (TGA, TAA orTAG) immediately after the LH_(N)/A coding sequence, unless it ispreferred to use a C-terminal tag to facilitate purification of thefusion protein, in which case no STOP codon would be inserted andtranslation would continue into the tag.

The entire DNA expression cassette is then cloned into, for example, avector suitable for expression of proteins in E. coli. Expression of theprotein is achieved by induction of transcription and the synthesizedprotein is isolated from the host cell by classical purificationtechniques.

Incorporation of an affinity tag would facilitate this last step.

The examples described above are purely illustrative of the invention.In synthesizing the agent the coupling of the TM to the modifiedclostridial neurotoxin or fragment thereof is achieved via chemicalcoupling using reagents and techniques known to those skilled in theart. Thus, although the examples given use exclusively the PDPH/EDAC andTraut's reagent chemistry any other coupling chemistry capable ofcovalently attaching the TM component of the agent to clostridialneurotoxin derived component and known to those skilled in the art iscovered by the scope of this application. Similarly it is evident tothose skilled in the art that either the DNA coding for either theentire agent or fragments of the agent could be readily constructed and,when expressed in an appropriate organism, could be used torecombinantly produce the agent or fragments of the agent. Such geneticconstructs of the agent of the invention obtained by techniques known tothose skilled in the art are also covered in the scope of thisinvention. TABLE 1 Possible Targeting Moieties (TM) Growth Factors: 1.Nerve growth factor (NGF); 2. Leukaemia inhibitory factor (LIF); 3.Basic fibroblast growth factor (bFGF); 4. Brain-derived neurotrophicfactor (BDNF); 5. Neurotrophin-3 (NT-3); 6. Hydra head activator peptide(HHAP); 7. Transforming growth factor 1 (TGF-1); 8. Transforming growthfactor 2 (TGF-2); 9. Transforming growth factor (TGF-); 10. Epidermalgrowth factor (EGF); 11. Ciliary neuro-trophic factor (CNTF).Cytokines: 1. Tumor necrosis factor (TNF-); 2. Interleukin-1 (IL-1); 3.Interleukin-1 (IL-1); 4. Interleukin-8 (IL-8). Peptides: 1. -Endorphin;2. Methionine-enkephalin; 3. D-Ala²-D-Leu⁵-enkephalin; 4. Bradykinin.Antibodies: 1. Antibodies against the lactoseries carbohydrate epitopesfound on the surface of dorsal root ganglion neurons (e.g. monoclonalantibodies 1B2 and LA4); 2. Antibodies against any of the receptors forthe ligands given above. 3. Antibodies against the surface expressedantigen Thyl (e.g. monoclonal antibody MRC OX7).

1. A non-cytotoxic agent that binds to a nociceptive afferent neuron,which comprises: a Targeting Moiety (TM) coupled to a modifiedclostridial neurotoxin in which the TM comprises a ligand to acell-surface binding site present on a nociceptive afferent neuron, andis capable of functionally interacting with a binding site causing aphysical association between the agent and the surface of a nociceptiveafferent neuron; and the heavy chain (H-chain) of the clostridialneurotoxin is removed or modified by chemical derivatisation, mutationor proteolysis to reduce or remove its native binding affinity for motorneurons; and the light chain (L-chain) of the clostridial neurotoxin ora fragment thereof retains a protease activity specific for componentsof the neurosecretory machinery; the TM and the modified H-chain, ifpresent, forming a molecule that introduces the L-chain or fragmentthereof into the cytosol of a nociceptive afferent neuron, therebyinhibiting the transmission of signals between a nociceptive afferentneuron and a projection neuron by controlling the release of at leastone neurotransmitter or neuromodulator from the nociceptive afferentneuron; and wherein the modified clostridial neurotoxin component is notobtained from tetanus neurotoxin.
 2. An agent according to claim 1,which comprises a Targeting Moiety (TM) coupled to a clostridialneurotoxin in which the H_(C) part of the H-chain is removed ormodified.
 3. An agent according to claim 1 in which the modified H-chainis the H_(N)-fragment of a clostridial neurotoxin.
 4. An agent accordingto claim 1 in which the clostridial neurotoxin component is obtainedfrom botulinum neurotoxin.
 5. An agent according to claim 4 in which theclostridial neurotoxin component is obtained from botulinum neurotoxinselected from the group consisting of botulinum neurotoxin type A,botulinum neurotoxin type B, and botulinum neurotoxin type C.
 6. Anagent according to claim 5, which is formed by the coupling of a TM tothe LH_(N) fragment of botulinum neurotoxin type A.
 7. An agentaccording to claim 5, which is formed by the coupling of a TM to theLH_(N) fragment of botulinum neurotoxin type B.
 8. An agent according toclaim 5, which is formed by the coupling of a TM to the LH_(N) fragmentof botulinum neurotoxin type C1.
 9. An agent according to claim 1 inwhich the H-chain is obtained from a different clostridial neurotoxinfrom that from which the L-chain is obtained.
 10. An agent according toclaim 9 in which the H-chain is obtained from botulinum neurotoxin typeA and the L-chain from botulinum neurotoxin type B.
 11. An agentaccording to claim 10, which is composed of a TM linked to the H_(N)fragment of botulinum neurotoxin type A and the L-chain from botulinumneurotoxin type B.
 12. An agent according to claim 1 in which theL-chain or L-chain fragment is linked to the H-chain by a directcovalent linkage.
 13. An agent according to claim 1 in which the L-chainor L-chain fragment is linked to the H-chain by a direct covalentlinkage that includes one or more spacer regions.
 14. An agent accordingto claim 1 in which the TM is capable of delivering the L-chain orL-chain fragment into the cytosol of a nociceptive afferent neuronunaided.
 15. An agent according to claim 1 in which the ability todeliver the L-chain or L-chain fragment into the cytosol of anociceptive afferent neuron is entirely contained within the TM.
 16. Anagent according to claim 1 in which the TM binds to a binding site thatis characteristic of a particular defined population of primarynociceptive afferent neurons.
 17. An agent according to claim 1 in whichthe TM binds to a binding site that undergoes retrograde transportwithin a nociceptive afferent neuron.
 18. An agent according to claim 1in which the TM comprises a ligand to a cell-surface receptor on anociceptive afferent neuron.
 19. An agent according to claim 18 in whichsaid receptor on a primary sensory afferent is selected from the groupconsisting of a growth factor receptor, a neuropeptide receptor, acytokine receptor, and a hormone receptor.
 20. An agent according toclaim 19 in which the TM comprises a ligand to a nerve growth factorreceptor.
 21. An agent according to claim 20 in which the TM comprisesnerve growth factor.
 22. An agent according to claim 21, which comprisesnerve growth factor linked to the LH_(N) fragment of botulinumneurotoxin type A.
 23. An agent according to claim 1 in which the TMcomprises a monoclonal antibody or is derived from a monoclonal antibodyto a surface antigen on a nociceptive afferent neuron.
 24. An agentaccording to claim 1 in which the TM is linked to the clostridialneurotoxin-derived component by a direct covalent linkage.
 25. An agentaccording to claim 1 in which the TM is linked to the clostridialneurotoxin-derived component by a direct covalent linkage that includesone or more spacer regions.
 26. An agent according to claim 1, whichprevents the release of a neurotransmitter or neuromodulator from anociceptive afferent neuron.
 27. An agent according to claim 1, whichbinds specifically to a nociceptive afferent neuron.
 28. An agentaccording to claim 1, wherein the TM is a growth factor.
 29. An agentaccording to claim 1, wherein the TM is a cytokine.
 30. An agentaccording to claim 1, wherein the TM is an antibody against alactoseries carbohydrate epitope found on the surface of dorsal rootganglion neurons.
 31. An agent according to claim 1, wherein the TM isan endorphin or endorphin receptor antibody.
 32. An agent according toclaim 1, wherein the TM is bradykinin or a bradykinin receptor antibody.33. An agent according to claim 1, wherein the TM is an enkephalin orenkephalin receptor antibody.
 34. A method for obtaining an agentaccording to claim 1, which comprises constructing a genetic constructthat codes for a modified clostridial neurotoxin or a fragment of aclostridial neurotoxin, incorporating said construct into a hostorganism, expressing the construct to produce the modified clostridialneurotoxin or fragment of a clostridial neurotoxin, and covalentlyattaching said clostridial neurotoxin or fragment thereof to a TM;wherein said modified clostridial neurotoxin or fragment of aclostridial neurotoxin is not obtained from tetanus neurotoxin.
 35. Amethod of controlling the release of a neurotransmitter orneuromodulator from a nociceptive afferent neuron by applying the agentof claim
 1. 36. A method according to claim 35, wherein said agent isinjected locally.
 37. A method according to claim 35, wherein said agentis injected spinally at the level of the spinal segment involved in theinnervation of an affected organ.
 38. A method of preventing oralleviating pain, which comprises administering an effective dose of theagent according to claim
 1. 39. A method according to claim 38, whereinsaid agent is injected locally.
 40. A method according to claim 38,wherein said agent is injected spinally at the level of the spinalsegment involved in the innervation of an affected organ.
 41. A methodfor preparing an agent according to claim 41 in the form of a fusionprotein, said method comprising expressing in a host organism a geneticconstruct which codes for the agent.
 42. A method according to claim 41,which further comprises constructing the genetic construct andtransforming the host with said construct.
 43. A method according toclaim 41, wherein the genetic construct includes a sequence encoding aspacer molecule by which the modified clostridial neurotoxin, or afragment thereof, is coupled to the TM.
 44. A method according to claim43, wherein the spacer molecule is selected from the group consisting of(SEQ ID NO:11) PPPIEGR, collagen-like spacer, and trypsin-sensitivediphtheria toxin peptide.
 45. A method according to claim 41, whereinthe nucleic acid sequence of the genetic construct is modified inaccordance with the codon bias of the host cell.
 46. A method accordingto claim 41, wherein the genetic construct incorporates a nucleic acidsequence encoding an affinity tag to facilitate purification of theassembled toxin.
 47. An agent that has been obtained in the form of afusion protein by the method according to claim 41.